Downloaded from rnajournal.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press

Comparison of mitochondrial and nucleolar RNase MRP reveals identical RNA components with distinct enzymatic activities and protein components

QIAOSHENG LU,1 SARA WIERZBICKI,1 ANDREY S. KRASLINIKOV,2 and MARK E. SCHMITT1 1Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, Syracuse, New York 13210, USA 2Department of Biochemistry and Molecular Biology, Penn State University, University Park, Pennsylvania 16802, USA

ABSTRACT RNase MRP is a ribonucleoprotein found in three cellular locations where distinct substrates are processed: the mitochondria, the nucleolus, and the cytoplasm. Cytoplasmic RNase MRP is the nucleolar enzyme that is transiently relocalized during . Nucleolar RNase MRP (NuMRP) was purified to homogeneity, and we extensively purified the mitochondrial RNase MRP (MtMRP) to a single RNA component identical to the NuMRP RNA. Although the protein components of the NuMRP were identified by mass spectrometry successfully, none of the known NuMRP proteins were found in the MtMRP preparation. Only trace amounts of the core NuMRP protein, Pop4, were detected in MtMRP by Western blot. In vitro activity of the two enzymes was compared. MtMRP cleaved only mitochondrial ORI5 substrate, while NuMRP cleaved all three substrates. However, the NuMRP enzyme cleaved the ORI5 substrate at sites different than the MtMRP enzyme. In addition, enzymatic differences in preferred ionic strength confirm these enzymes as distinct entities. Magnesium was found to be essential to both enzymes. We tested a number of reported inhibitors including puromycin, pentamidine, lithium, and pAp. Puromycin inhibition suggested that it binds directly to the MRP RNA, reaffirming the role of the RNA component in catalysis. In conclusion, our study confirms that the NuMRP and MtMRP enzymes are distinct entities with differing activities and protein components but a common RNA subunit, suggesting that the RNA must be playing a crucial role in catalytic activity. Keywords: RNase MRP; ribonucleoprotein; endoribonuclease; mitochondrial DNA replication; rRNA processing;

INTRODUCTION A3 site, leading to the generation of the mature 59-end of the 5.8S rRNA (Schmitt and Clayton 1993; Chu et al. 1994; RNase mitochondrial RNA processing (RNase MRP) is a Lygerou et al. 1996; Cai et al. 2002). ribonucleoprotein endoribonuclease. RNase MRP was ini- During mitosis in S. cerevisiae, RNase MRP localizes to tially isolated from mouse cell mitochondria, as an in vitro temporal asymmetric MRP (TAM) bodies where it is enzymatic activity that cleaves RNA transcripts comple- believed to cleave the mRNA for a B-type cyclin (Clb2) mentary to the origin of replication consistent with it, to promote the end of mitosis (Cai et al. 2002; Gill et al. forming the RNA primers for the leading strand of mito- 2006). in the for the RNA component of chondrial DNA replication (Chang and Clayton 1987; human RNase MRP cause the autosomal recessive genetic Chang et al. 1987). Despite its mitochondrial function, disease cartilage hair hypoplasia (Ridanpa¨a¨ et al. 2001), the majority of RNase MRP is found in the nucleolus which is characterized by short-limb dwarfism, brittle (Reimer et al. 1988; Gold et al. 1989). In Saccharomyces sparse hair, and immunodeficiency. In addition, human cerevisiae, nucleolar RNase MRP (NuMRP) was shown to RNase MRP mutations lead to changes in cyclin mRNA play a direct role in processing the rRNA precursor at the abundance and rRNA processing (Thiel et al. 2005, 2007). The composition of the NuMRP has been well-estab- Reprint requests to: Mark E. Schmitt, Department of Biochemistry and lished in S. cerevisiae where a complex containing a single Molecular Biology, State University of New York Upstate Medical RNA and at least 10 protein components has been iden- University, 750 East Adams Street, Syracuse, NY 13210, USA; e-mail: tified (Schmitt and Clayton 1992; Salinas et al. 2005). Eight [email protected]; fax: (315) 464-8750. Article published online ahead of print. Article and publication date of the proteins (Pop1p, Pop3p, Pop4p, Pop5p, Pop6p, are at http://www.rnajournal.org/cgi/doi/10.1261/rna.1893710. Pop7p, Pop8p, and Rpp1p) are shared with the yeast

RNA (2010), 16:00–00. Published by Cold Spring Harbor Laboratory Press. Copyright Ó 2010 RNA Society. 1 Downloaded from rnajournal.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press

Lu et al. nucleolar RNase P (Chamberlain et al. 1998), a closely into 15 fractions. The RNA levels in each fraction were related ribonucleoprotein endoribonuclease that has mul- analyzed by Northern analysis (Fig. 1). The RNA profiles tiple activities in the cell, including processing cytoplas- were similar for both of the RNase MRP enzymes. The mic tRNA precursors to generate mature 59-termini. Two RNA peak was broader and consistently one fraction later unique protein components of NuMRP are Snm1p and in the MtMRP, but the difference was not significant. This Rmp1p. Snm1p and Rmp1p bind to the RNase MRP RNA indicates that the density of the two complexes is slightly but not the RNase P RNA (Schmitt and Clayton 1994; different but comparable (Fig. 1). Salinas et al. 2005). The RNA component of RNase MRP is Proteins precipitated from both MtMRP and NuMRP structurally related to the RNA component of RNase P samples containing the same amount of the MRP RNA (Chamberlain et al. 1998). Though the RNase P RNA has were separated by SDS-PAGE, and visualized by MALDI been shown to be catalytically active, this has never been compatible silver stain (Fig. 2A). The TAP-tag purified demonstrated for the RNase MRP RNA (Guerrier-Takada NuMRP prior to the glycerol gradient served as a control. et al. 1983; Stohl and Clayton 1992; Pannucci et al. 1999; Individual protein bands were isolated, digested in-gel by Kikovska et al. 2007). trypsin, and analyzed by mass spectrometry. All 10 of the Several in vitro assays have been established to test previously identified NuMRP proteins were visualized and RNase MRP cleavage activity in S. cerevisiae which include identified successfully in the control (Fig. 2A, lane 1). In the characterized in vitro substrates for all three compartments NuMRP fraction isolated by glycerol gradient centrifuga- (Stohl and Clayton 1992; Venema and Tollervey 1999; Cai tion, the protein concentration was much lower, and only and Schmitt 2001; Gill et al. 2004). With the availability of six of the 10 proteins could be visualized and five of these highly purified NuMRP and partially purified mitochon- proteins were in a concentration high enough to be iden- drial RNase MRP (MtMRP) we examined the biochemical tified by mass spectrometry as Pop1p, Rpp1p, Pop5p, characteristics and enzymatic behavior of these two en- zymes. The results strongly support the hypothesis that NuMRP and MtMRP are different identities that possess a common enzymatic RNA but distinct protein compo- nents and enzymatic activities.

RESULTS

Protein composition analysis of MtMRP and NuMRP preparations The S. cerevisiae NuMRP was purified to homogeneity using a tagged version of Pop4 and a modified tandem affinity purification (TAP) technique (see Materials and Methods). The NuMRP preparation contained a single RNA of 340 nucleotides (nt) encoded by the yeast NME1 gene, and 10 proteins (Salinas et al. 2005). The MtMRP was partially purified by conventional ion exchange chro- matography and glycerol density-gradient centrifugation. Throughout the purification the enzyme was followed us- ing cleavage of the mitochondrial substrate as an assay (Stohl and Clayton 1992; Cai and Schmitt 2001). The final preparation contained a single RNA of 340 nt that was the same RNA in the NuMRP preparation. Since both NuMRP and MtMRP preparations contained an identical RNA component, RNA levels were used to quantitate amounts of the two enzymes and allow direct comparison. Differences in protein and RNA composition can lead to differences in the density of a ribonucleoprotein complex and a difference in its behavior by density gradient cen- FIGURE 1. Glycerol gradient centrifugation of NuMRP and MtMRP. trifugation. The purified RNase MRP preparations, each Purified NuMRP or MtMRP containing 50–80 ng of the MRP RNA containing the same amount of the MRP RNA, were were centrifuged in parallel on a 15%–30% glycerol density-gradient, and fractioned as described in the Material and Methods. RNA analyzed in parallel in a 15%–30% glycerol density-gradi- isolated from the gradient fractions and the MRP RNA was detected ent. Gradients were centrifuged together and then divided by Northern analysis. (A) MtMRP. (B) NuMRP.

2 RNA, Vol. 16, No. 3 Downloaded from rnajournal.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press

Enzymatic differences of RNase MRP

compared in vitro on three different substrates: the rRNA substrate, the CLB2 substrate, and the mitochondrial ORI5 substrate. The MtMRP cleaves the mitochondrial ORI5 substrate at the 59-end of the GC cluster C/CSB II region, leaving a 39-fragment of z102 nt; this is consistent with the reported cleavage pattern (Fig. 3A; Stohl and Clayton 1992; Cai and Schmitt 2001). However, MtMRP was able to cleave neither the rRNA substrate nor the CLB2 substrate when tested under a variety of conditions (Fig. 3B,C). NuMRP cleaved the 147-nt rRNA substrate at the A3 site, leaving a 67-nt 39-fragment. When NuMRP was incubated with the 268-nt CLB2 substrate, multiple cleavage sites were FIGURE 2. Protein analysis of NuMRP and MtMRP. (A) Protein samples were separated on a 7.5%–17.5% SDS-PAGE gel and seen, consistent with previous reports (Gill et al. 2004). visualized using MALDI compatible silver staining. Protein compo- NuMRP recognizes mitochondrial ORI5 substrate; how- nents were excised, digested by trypsin, and analyzed by MALDI-TOF ever, it cleaves the mitochondrial substrate at multiple mass spectrometry (see Materials and Methods). M, molecular-weight positions different than those cleaved by MtMRP, creating markers. (Lane 1)10mL of TAP-tag purified NuMRP prior to glycerol gradient. All 10 NuMRP protein components were confirmed in this five major fragments on the gel. There is no corresponding preparation (not shown). (Lane 2) Precipitated proteins from MtMRP 102-nt fragment generated by NuMRP even at lower containing 1000 picograms (pg) of the MRP RNA. (Lane 3) Pre- enzyme concentrations (Fig. 4A). When MtMRP is mixed cipitated proteins from NuMRP containing 1000 pg of the MRP RNA. with NuMRP at various ratios, the mixed enzymes cleaved Five of the 10 proteins successfully identified by mass spectrometry of this sample are indicated. (B) Proteins precipitated from both MtMRP the mitochondrial substrate at both the MtMRP cleavage and NuMRP containing 1000 pg of the MRP RNA were separated on site and NuMRP cleavage sites. Indeed, cleavage at the a 12.5% SDS-PAGE gel, transferred to nylon membrane, and Pop4 mitochondrial site seems to preclude further cleavage by was detected using anti-TAP antibody. Both preparations were made the NuMRP (Fig. 3D). from the same yeast strain expressing a Pop4:TAP fusion as its only source of Pop4 protein. Part of the protein is specifically cleaved during the NuMRP purification giving a smaller protein, but leaving the antibody epitope. The arrow indicates the size of the full-length Titration and time course on substrates uncleaved protein in the MtMRP preparation. We also compared the two enzymes to see if their rate of catalysis was the same and to see if they would demonstrate Pop6p, and Pop8p (Fig. 2A, lane 3). In the MtMRP fraction comparable cleavages under higher enzyme concentrations most of the major proteins have been successfully identified and longer reaction times. To compare the cleavage abilities by mass spectrometry after in-gel trypsin digestion (Table 1). of MtMRP and NuMRP, both enzymes were serially diluted None of the protein components of the NuMRP were and incubated with substrate. Minimal cleavage activity found in the MtMRP preparations (Fig. 2A, lane 2). Hence, (some detectable cleavage product) was seen in prepara- none of the 10 NuMRP protein components appear to be tions containing 78 femtograms (fg) of RNase MRP RNA associated with the MtMRP preparation. Table 1 provides for MtMRP, and 39 fg of RNase MRP RNA for NuMRP. a summary of potential MtMRP proteins identified in Quantitation of the amount of total products generated per MtMRP fractions. Confirmation of any of these as genuine fg of RNase MRP RNA (see Materials and Methods) MtMRP proteins still requires further experimentation. indicated that NuMRP was about four times more active Proteins precipitated by TCA from both MtMRP and than the MtMRP enzyme (Fig. 4A). This is possibly the NuMRP samples with the same amounts of MRP RNA result of a partial inactivation during the purification process were analyzed by Western blot with anti-TAP-tag antibody or the innate ability of the NuMRP to process more (Fig. 2B); while Pop4 is clearly visible in NuMRP preparation, efficiently. The MtMRP purification is more complicated, only a trace amount of Pop4 was detected in MtMRP (arrow but not considerably harsher than the NuMRP purification. in Fig. 2B). This result indicates that Pop4 is clearly not a The RNA cleavage activity of both enzymes was time- protein component of MtMRP. A similar result was seen with dependent. The cleavage products could be seen starting an anti-peptide antibody against Rpp1 (data not shown). from 1 min of incubation for NuMRP and 5 min for MtMRP. The concentration of cleavage products increased with the incubation time, and saturated after 10–30 min Comparison of enzymatic cleavage on different for NuMRP and MtMRP (Fig. 4B, and data not shown). substrates With both enzymes, even at the highest enzyme concen- Since the two enzymes appeared to have very different trations and the longer reaction times, different cleavage protein compositions, we examined the differences of both products were observed. This result indicates that the nucleolar and MtMRP in substrate recognition and cleav- NuMRP and MtMRP are well separated and that they are age. The catalytic activities of the two enzymes were distinct enzymes.

www.rnajournal.org 3 Downloaded from rnajournal.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press

Lu et al.

TABLE 1. Potential RNase MRP proteins that were identified in the MtMRP preparation

ORF name Gene Description

YOR356W CIR2 Probable electron transfer flavoproteinubiquinone oxidoreductase, mitochondrial precursor YNL073W MSK1 Lysyl-tRNA synthetase, mitochondrial YPL104W MSD1 Aspartyl-tRNA synthetase, mitochondrial YGL078C DBP3 Probable ATP-dependent RNA helicase YDR148C KGD2 Dehydrolipoyllysine residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex, mitochondrial precursor YHR011W DIA4 Seryl-tRNA synthetase, mitochondrial YMR192W GYL1 Putatative GTPase activating protein YOR091W TMA46 Associated w/ ribosomes YPL097W MSY1 Tyrosine-tRNA ligase activity YCR003W MRPL32 Structural constituent of ribosome, mitochondrial YDL197C ASF2 Molecular_function unknown YGL131C SNT2 molecular_function unknown YHR150W PEX28 Molecular_function unknown YGR155W CYS4 Cystathionine b-synthase activity YHR127W HSN1 Molecular_function unknown YMR129W POM152 Nuclear Pore protein YNL005C MRP7 Structural constituent of ribosome, mitochondrial YBR111C YSA1 Phosphoribosyl-ATP diphosphatase activity YHL010C ETP1 Molecular_function unknown YBL064C PRX1 Thioredoxin peroxidase activity YBR263W SHM1 Glycine hydroxymethyltransferase activity YDL027C YDL027C Molecular_function unknown, mitochondrial YGL228W SHE10 Molecular_function unknown YGR085C RPL11B Structural constituent of ribosome YJR030C YJR030C Molecular_function unknown YOR096W RPS7A Structural constituent of ribosome YFR006W YFR006W X-Pro aminopeptidase activity YJL209W CBP1 mRNA binding YGR086C PIL1 Phosporylated protein YNL071W LAT1 Dihydrolipoyllysine-residue acetyltransferase activity YPL004C LSP1 Protein kinase inhibitor activity

Bands were excised from the gel, trypsin digested, and the resulting peptides were analyzed by mass spectrometry as described in Materials and Methods. Proteins of known function that are not localized to the mitochondria were excluded as probable contaminants. Gene descriptions are from the Saccharomyces Genome Database (Hong et al. 2008).

Salt sensitivity et al. 1997), though it has never been shown directly in an in vitro assay. We examined whether lithium would inhibit Differences in protein composition between the two en- our in vitro RNase MRP assays and whether or not the zymes may also lead to differences in optimal cleavage different enzymes might display different sensitivities. In conditions. To examine this possibility we assayed MtMRP our assays, Li+ does not significantly inhibit MtMRP on mitochondrial ORI5 substrate and NuMRP on all three cleavage of ORI5 substrate at 100 mM or less concentra- substrates at a variety of potassium and magnesium tion, and does not significantly inhibit NuMRP cleavage of concentrations. The MtMRP activity on the mitochondrial ORI5 substrate at 50 mM or less concentration (Fig. 6). substrate was both K+ and Mg2+ dependent, with the best This inhibition pattern is similar to the inhibition of RNase cleavage activity observed at 75 mM K+ and 10 mM Mg2+ MRP activity in high K+ buffer, which indicates that at least (Fig. 5). In contrast, NuMRP activity preferred no or low in vitro inhibition by Li+ is caused by changes in the ionic salt concentrations. Although Mg2+ is necessary, NuMRP strength of the assay buffer as opposed to specific inhibition catalytic activity is relatively stable in a broad range of Mg2+ of the enzyme (Fig. 6). concentrations, and K+ is not essential to NuMRP. This was found to be true for cleavage on all three substrates, in- Inhibitors of RNase MRP dicating an enzyme preference as opposed to changes in substrate structure. Adenosine 39,59-diphosphate (pAp) has been shown to be It has been reported that lithium directly inhibits RNase an inhibitor of a number of including the MRP cleavage at the A3 site of pre-rRNA in vivo (Dichtl 59/39 exonucleases Xrn1p and Rat1p (Dichtl et al. 1997).

4 RNA, Vol. 16, No. 3 Downloaded from rnajournal.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press

Enzymatic differences of RNase MRP

and mouse MtMRP (Vioque 1989; Potuschak et al. 1993). In our in vitro experiment, 6 mM puromycin inhibited approximately half of ORI5 substrate cleavage by MtMRP, and half of the ORI5 substrate cleavage activities by NuMRP were inhibited at 8 mM puro- mycin (Table 2). This is a concentration 1000-fold higher than that found to in- hibit the ribosome (Starck and Roberts 2002), and equivalent to what was seen for the mouse RNase MRP and P en- zymes (Potuschak et al. 1993). This con- firms puromycin as an inhibitor of RNase MRP but revealed no significant enzymatic differences between the two enzymes. However, similar levels of in- FIGURE 3. In vitro cleavage assays of RNase MRP on three different substrates. RNA hibition of both enzymes by puromycin 32 substrates 39-end labeled with P were incubated with either NuMRP or MtMRP for 30 min at indicate that it binds the RNA compo- 37°C. The cleavage reactions were analyzed on a 6% polyacrylamide, 7 M urea gel as described in Materials and Methods. (A) Mitochondrial Ori5 substrate. (B) rRNA A3 substrate. (C) nent in order to inhibit activity, exem- CLB2 mRNA substrate, (D) MtMRP and NuMRP were mixed together on the mitochondrial plifying the role of the RNA in catalytic substrate at varying ratios. activity. Pentamidine, an RNA binding inhib- Xrn1 degrades the CLB2 mRNA after RNase MRP specif- itor of some ribonucleoproteins, was also tested (Sands ically cleaves its 59-untranslated region (UTR). Our in vitro et al. 1985; Liu et al. 1994; Sun and Zhang 2008). Depending experiments showed that pAp did not inhibit RNase MRP on whether pentamidine binds to the RNA component of cleavage activities on any of the three substrates (data not RNase MRP or the RNA substrates, it may have a different show). inhibitory effect on the mitochondrial and NuMRP. Our Puromycin, an antibiotic that inhibits protein synthe- results showed that pentamidine inhibits substrates cleav- sis, has been previously reported to inhibit mouse RNase P age activities of both MtMRP and NuMRP. Pentamidine

FIGURE 4. Enzyme titration and time course of RNase MRP cleavage by NuMRP and MtMRP. (A) The 32P-labeled mitochondrial substrate was incubated with increasing amounts of MtMRP or NuMRP for 30 min. Enzyme levels were quantitated by the MRP RNA levels, which are indicated. (B) The 32P-labeled mitochondrial substrate was incubated with the MtMRP or NuMRP for increasing amounts of time. The cleavage reactions were analyzed on a 6% polyacrylamide, 7 M urea gel as described in Materials and Methods.

www.rnajournal.org 5 Downloaded from rnajournal.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press

Lu et al.

to mitochondria or have a role in mitochondrial metabo- lism (Table 1). This was expected since the purification starts with a mitochondrial fractionation. Some of these proteins are known to be RNA binding proteins and may be genuine components of the MtMRP complex, though none of these have been directly confirmed yet as genuine components. Western blot analysis on both NuMRP and MtMRP preparation with the same amount of RNase MRP RNA showed only a trace amount of Pop4 protein in MtMRP, <1% of the amount in NuMRP. Pop4 is a core component of the NuMRP and the nuclear RNase P enzymes. Indeed, addition of this protein is critical for reconstitution of the human RNase P activity (Mann et al. 2003). These results indicate that the protein compositions of NuMRP and MtMRP are different despite sharing the same RNA compo- nent. There are no reports of a mitochondrial RNA process- ing defect associated with any of the known NuMRP protein FIGURE 5. Effect of ionic strength on NuMRP and MtMRP activity. components. Indeed, our laboratory has directly looked with The 32P-labeled mitochondrial ORI5 substrate was incubated with both NuMRP-specific proteins, Snm1 and Rmp1, and has nuMRP and mtMRP for 30 min in buffers with different concentra- found nothing (Cai et al. 1999; K Salinas and ME Schmitt, tions of K+ or Mg2+. Except for the indicated ion, standard reaction conditions were used. The cleavage reactions were analyzed on a 6% unpubl.). Though it is a negative result, it is still consistent polyacrylamide, 7 M urea gel as described in Materials and Methods. with our findings in this paper. Interestingly, the RNase MRP RNA from a number of without mitochondria, inhibits about half of ORI5 cleavage activities of both en- such as the microsporidia, Encephalitozoon cuniculi, are zymes at the concentration around 100 mM; however, more than twice that amount is needed to inhibit the NuMRP from cleaving the rRNA substrate (Table 2). These results suggest that pentamidine binds to the substrate RNA to inhibit RNase MRP function.

DISCUSSION RNase MRP is a site-specific ribonucleoprotein endoribo- with three distinct functions. These include cleaving RNA transcripts to generate primers for DNA replication in mitochondria (Chang and Clayton 1987; Chang et al. 1987), processing the A3 site of the rRNA precursor to form the 5.8S(S) ribosomal RNA in the nucleolus (Chu et al. 1994; Lygerou et al. 1996), and cleaving the 59-UTR of CLB2 mRNA, which promotes its degradation at the end of mitosis (Gill et al. 2006). In S. cerevisiae, NuMRP has been well studied and it contains a single RNA and 10 protein components. However, little is known about the composition of the MtMRP, except that it has the same RNA component as the NuMRP. In this study, MtMRP was purified to a single RNA component using conventional biochemical techniques. When analyzed in parallel by glycerol density gradient centrifugation, MtMRP consistently peaked at a slightly lower density fraction, suggesting its density is less than FIGURE 6. Effect of lithium on RNase MRP catalytic activities. (A) that of NuMRP. Interestingly, the MtMRP fractions did not The 32P-labeled substrate was incubated for 30 min with NuMRP or contain any of the protein components of NuMRP. Indeed, MtMRP under standard reaction conditions with increasing concen- trations of lithium and either the ORI5 or A3 substrates. (B) Effect of we identified >50 proteins in these samples. More than lithium or potassium on cleavage of the A3 substrate by NuMRP. The 60% of the identified proteins were known to be localized cleavage activity without adding lithium or potassium is set as 100%.

6 RNA, Vol. 16, No. 3 Downloaded from rnajournal.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press

Enzymatic differences of RNase MRP

a known inhibitor both in vitro and in TABLE 2. Summary of results of various inhibitors on RNase MRP activity vivo of the Xrn1 and Rat1 exonucleases Ori5 substrate A3 substrate (Dichtl et al. 1997). We found that pAp has no direct inhibition on RNase MRP Inhibitor Ki MtMRP Ki NuMRP Ki NuMRP activity in our in vitro assay. However Lithium 100 mM 100 mM 50 mM the Xrn1 and Rat1 exonucleases degrade Potassium <25 mM; >100 mM >75 mM >50 mM pAp None up to 10 mM None up to 10 mM None up to 10 mM RNase MRP cleavage products in vivo, Puromycin 6 mM 8 mM 6 mM inhibition of RNase MRP may be the Pentamidine 100 mM 100 mM 250 mM indirect result of product inhibition. Indeed, we have seen strong product K is the concentration of the compound where z50% of the RNase MRP activity remains. i inhibition in our in vitro assays (data not shown). In vivo, this product in- missing a number of the conserved helices in what is hibition must also be strong since reduction of cellular pAp believed to be the substrate specificity (Woodhams by including methionine in the yeast media still leads to the et al. 2007). This suggests that these may be binding accumulation of the RNase MRP rRNA substrate in the platforms for mitochondrial specific proteins. presence of Li+ (Dichtl et al. 1997). So far, three in vitro substrates of RNase MRP have been Several lines of evidence from this study indicate that the established in S. cerevisiae: the ORI5 substrate for the MtMRP and NuMRP are distinct entities that contain the MtMRP (Cai and Schmitt 2001) and the A3 and CLB2 same RNA component. First the two enzymes have differ- substrates for the NuMRP (Gill et al. 2004). In our results, ent protein components in their preparation. Second, the MtMRP enzyme cleaves only the mitochondrial substrate two enzymes have distinct substrate specificities and dis- while the NuMRP enzyme cleaves all three substrates. tinct activities on common substrates. Third the two en- However, NuMRP cleaves the ORI5 substrate at multiple zymes have different ionic strength optima for catalysis, sites, different from MtMRP cleavage, generating five major even on the same substrate. Despite these differences, both fragments on the gel. In vitro cleavage study with mouse enzymes contain the same RNA component and are endo- NuMRP on the mitochondrial substrate was reported pre- . Both enzymes require magnesium and are viously (Karwan et al. 1991). In this particular case, the inhibited by similar concentrations of puromycin. The dif- nucleolar enzyme was found to produce two additional ferences and similarities indicate that the RNA component cleavages to the one that was performed by the mitochon- is the catalytic center that is shared by both enzymes. Roles drial enzyme. Indeed, the mouse MtMRP may also have of the protein components may be to promote the active a very different protein composition than the NuMRP. structure of the RNA component, ensure its proper lo- NuMRP and MtMRP were found to have different salt calization, and help provide substrate specificity. sensitivities. Potassium is essential to MtMRP cleavage activity with the optimum being at 75 mM. However, MATERIALS AND METHODS NuMRP cleaves ORI5, A3, and CLB2 substrate at highest efficiency without potassium in the reaction buffer and Strains and media even shows partial inhibition at 75 mM KCl. These dif- ferences indicate distinct enzymatic difference between the Yeast media and genetic manipulations have been described pre- enzymes and not changes in substrate folding. viously (Gill et al. 2004; Salinas et al. 2005). The YSW1 strain which Lithium has been shown to directly inhibit cleavage at contains a TAP fusion cassette inserted into the carboxy-terminal of T T the A3 site of the rRNA precursor in vivo (Dichtl et al. the POP4 gene has the genotype MATa POP4 TAPTAG TRP1ks pep4:LEU2 nuc1TLEU2 sep1TURA3 trp-1his3–11,15 can-100 1997). Since RNase MRP is responsible for that cleavage ura3–1 leu2–3,112 and was used for both NuMRP and MtMRP event it has been proposed that the enzyme is sensitive to purification. lithium. Our in vitro experiments showed both NuMRP + and MtMRP cleavage activity were decreased in high Li RNase MRP purification concentrations. NuMRP cleavage activity on the rRNA substrate was inhibited >60% in 50 mM Li+, and catalytic NuMRP was purified from strain YSW1 using a modified TAP pro- activity was completely inhibited when the Li+ concentra- tocol. Briefly, YSW1 cells were resuspended in buffer Z-150 (20 mM tion was >100 mM. However, variations in K+ concentra- Tris-HCl [pH 8.0], 150 mM KCl, 1 mM EDTA, 10% [vol/vol] glycerol, 1 mM dithiothreitol, 1 mM phenylmethylsufonyl fluo- tion resulted in a similar pattern of inhibition. We + ride) and were disrupted by passing through a Microfluidics conclude that Li inhibition of nuclear RNase MRP activity M-110L microfluidizer (Microfluidics) eight to 10 times. Broken in vitro is linked to high monovalent ion concentration cells were removed at 10,000g for 15 min, and the cell extract was + and not a specific inhibition. It is known that high Li clarified by centrifugation at 100,000g for 100 min. The clarified concentrations directly result in inhibition of the MET22 extract was loaded onto a UNOspere Q (Bio-Rad) column gene product which is responsible for breaking down pAp, equilibrated with buffer Z-150. The column was washed in four

www.rnajournal.org 7 Downloaded from rnajournal.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press

Lu et al.

column volumes of buffer Z-150 and proteins were eluted with Northern analysis when the RNA concentration was low as buffer Z-350 (20 mM Tris-HCl [pH 8.0], 350 mM KCl, 1 mM described previously (Cai et al. 1999, 2002). The probe used for EDTA, 10% [vol/vol] glycerol, 1 mM dithiothreitol, 1 mM Northern analysis was a 440-base pair PCR fragment of NME1 phenylmethylsufonyl fluoride). The eluted fraction was then used DNA. The fluorescence intensity of ethidium bromide stained for NuMRP purification using a TAP protocol previously de- RNA or the intensity of the Northern blot hybridization signals scribed in detail (Gill et al. 2004). Addition of the UNOspere were captured on a Typhoon-9410 Imager (GE Healthcare Bio- Q column removed any residual RNase P that may contaminate Sciences). RNA concentrations were calculated using ImageQuant the purification (Lygerou et al. 1996). 5.0 software (GE Healthcare). The known concentration of RNA MtMRP was purified from the same strain that was used for transcripts generated from pMES206 served as a control NuMRP preparation. The yeast strain YSW1 was grown in 12 L of and measure for quantitation. from this plasmid YPD at 30°Cto23 108 cells/mL with vigorous aeration. Cells using the SP6 RNA polymerase can generate a nearly exact were harvested by centrifugation and washed with water. The cells duplication of the endogenous RNase MRP RNA, except for the were preincubated in three volumes of zymolyase preincubation addition of a guanosine at the 59-end and seven fewer nucleotides buffer (0.1 M Tris-HCl [pH 9.3], 5 mM EDTA, 10 mM DTT) at at the 39-end. 30°C for 30 min. Collected cells were washed with three volumes of zymolyase digestion buffer (0.02 M sodium phosphate [pH In vitro RNA cleavage assay 7.4], 1.2 M sorbitol, 5 mM EDTA, 1 mM DTT), and then incubated in three volumes of zymolyase buffer with zymolyase RNA substrate preparation, 39-end labeling, and in vitro RNA 100T at 1 mg per mL of original packed cells at 30°C with gentle cleavage assays were carried out as previously described (Cai and agitation. The spheroplasts were washed three times with three Schmitt 2001; Gill et al. 2004). The 145-nt transcript from plasmid volumes of cold 1.2 M sorbitol-1 mM EDTA and resuspended in p64/HS40-ori5/4.4 served as the mitochondrial substrate (Cai and one volume of cold breaking buffer (10 mM Tris-HCl [pH7.5], Schmitt 2001), the 147-nt transcript of pJA110 containing the A3 0.6 M Sorbitol, 1 mM EDTA, and 0.5 mM PMSF). Spheroplasts site served as the rRNA substrate, and the 268-nt RNA transcript were broken in a Dounce homogenizer using 15 strokes, and cell of pJA108 containing the 59-UTR of CLB2 served as CLB2 sub- debris was removed by centrifugation at 3000g for 5 min. strate (Gill et al. 2004). The results of all experiments were cap- Mitochondria were pelleted by centrifugation of the supernatant tured on a Typhoon-9410 Imager (GE Healthcare Bio-Sciences). at 18,000 rpm for 15 min in a Beckman JA-20 rotor. The mi- This allowed for accurate quantitation of cleavage products. When tochondrial pellets were resuspended in breaking buffer, and the more than one product was observed, the signal corresponding to low-speed/high-speed spin cycle was repeated three more times. all of the products was summed together to determine the total Mitochondrial pellets were resuspended at 10–20 mg/mL protein amount of product. in mitochondria lysate buffer (20 mM Tris-HCl [pH 8.0], 1 mM EDTA, 1% Triton X-100, 0.2 M KCl, 10% glycerol, 1 mM PMSF, 1 mM DTT). The mitochondria were lysed by 15 strokes in a glass Protein analysis and identification Dounce homogenizer. Mitochondrial crude extracts were dialyzed Proteins in RNase MRP samples containing z8 ng of RNase MRP for 2 h against 2 L of buffer A-100 (50 mM Tris-HCl [pH 8.0], RNA were precipitated with the addition of trichloroacetic acid 100 mM NaCl, 5 mM EDTA, 10% Glycerol, 0.1% [v/v] Tween 20, (Brown et al. 1989). The pellets were resuspended in 15 mLof 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol). The loading buffer (62.5 mM Tris-HCl [pH 6.8], 2% sodium dodecyl cell extract was clarified by centrifugation at 100,000g for 100 min. sulfate [SDS], 10% glycerol, 0.1 M DTT, and 0.01% bromophenol Ion-exchange chromatography with DEAE-Sephacel (GE Health- blue), and proteins were separated on a 7.5%–17.5% gradient care Bio-Sciences) and Bio-Rex 70 (Bio-Rad) were performed. SDS-PAGE gel. Proteins were stained using a MALDI compatible Eluate of Bio-Rex 70 was concentrated with a Centricon YM-100 silver stain (Mortz et al. 2001). Individual protein bands were ultrafiltration device, and could be stored at À70°C. The concen- excised from the gel using a fresh razor blade, the gel pieces were trated eluate from the Bio-Rex 70 was applied on a glycerol digested with trypsin and subjected to MADI-TOF mass spec- gradient (see below). The peak fractions with RNase MRP RNA trometry (Shevchenko et al. 1996). Mass spectrometry data were were pooled for further analysis (Glick and Pon 1995; Cai and collected at the Proteomics Core Facilities at the State University Schmitt 2001; Boldogh and Pon 2007). of New York Upstate Medical University using a TOF Spec 2E mass spectrometer (Waters Corp.) or at the State University of Glycerol gradient centrifugation New York at Oswego, Department of Biological Science using an AutoFlex II (Bruker BioSciences). For glycerol gradient centrifugation, 200 mL of the concentrated Bio-Rex70 eluate were loaded onto 15%–30% glycerol gradients in 20 mM Tris-HCl (pH 8.0), 50 mM KCI, 0.5 mM EDTA, and ACKNOWLEDGMENTS 1 mM DTT, and centrifuged for 13.5 h at 38,000 rpm in a Beckman SW55Ti rotor. Gradients were fractionated from the We thank K. Salinas for comments on the manuscript and for bottom using a peristaltic pump. helpful discussions during this investigation. This work was supported by grants GM063798 and GM064634 from the National RNA analysis and quantitation Institute of General Medical Sciences to M.E.S., and by grant GM085149 to A.S.K. RNase MRP RNA was analyzed by denaturing polyacrylamide gel electrophoresis followed by ethidium bromide staining or by Received August 19, 2009; accepted November 20, 2009.

8 RNA, Vol. 16, No. 3 Downloaded from rnajournal.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press

Enzymatic differences of RNase MRP

REFERENCES a primordial catalytic ribonucleoprotein in RNA-based catalysis. Mol Cell 12: 925–935. Boldogh IR, Pon LA. 2007. Purification and subfractionation of Mortz E, Krogh TN, Vorum H, Gorg A. 2001. Improved silver mitochondria from the yeast Saccharomyces cerevisiae. Methods staining protocols for high sensitivity protein identification using Cell Biol 80: 45–64. matrix-assisted laser desorption/ionization-time of flight analysis. Brown RE, Jarvis KL, Hyland KJ. 1989. Protein measurement using Proteomics 1: 1359–1363. bicinchoninic acid: Elimination of interfering substances. Anal Pannucci JA, Haas ES, Hall TA, Harris JK, Brown JW. 1999. RNase Biochem 180: 136–139. P from some Archaea are catalytically active. Proc Natl Acad Cai T, Schmitt ME. 2001. Characterization of MRP Sci 96: 7803–7808. function. Methods Enzymol 342: 135–142. Potuschak T, Rossmanith W, Karwan R. 1993. RNase MRP and RNase Cai T, Reilly TR, Cerio M, Schmitt ME. 1999. Mutagenesis of SNM1, P share a common substrate. Nucleic Acids Res 21: 3239–3243. which encodes a protein component of the yeast RNase MRP, Reimer G, Raska I, Scheer U, Tan EM. 1988. Immunolocalization of reveals a role for this ribonucleoprotein endoribonuclease in 7-2-ribonucleoprotein in the granular component of the nucleo- plasmid segregation. Mol Cell Biol 19: 7857–7869. lus. Exp Cell Res 176: 117–128. Cai T, Aulds J, Gill T, Cerio M, Schmitt ME. 2002. The Saccharomyces Ridanpa¨a¨ M, van Eenennaam H, Pelin K, Chadwick R, Johnson C, cerevisiae RNase mitochondrial RNA processing is critical for cell Yuan B, vanVenrooij W, Pruijn G, Salmela R, Rockas S, et al. 2001. cycle progression at the end of mitosis. Genetics 161: 1029–1042. Mutations in the RNA component of RNase MRP cause a pleio- Chamberlain JR, Lee Y, Lane WS, Engelke DR. 1998. Purification and tropic human disease, cartilage–hair hypoplasia. Cell 104: 195– characterization of the nuclear RNase P holoenzyme complex 203. reveals extensive subunit overlap with RNase MRP. & Dev Salinas K, Wierzbicki S, Zhou L, Schmitt ME. 2005. Characteriza- 12: 1678–1690. tion and purification of Saccharomyces cerevisiae RNase MRP re- Chang DD, Clayton DA. 1987. A mammalian mitochondrial RNA veals a new unique protein component. J Biol Chem 280: 11352– processing activity contains nucleus-encoded RNA. Science 235: 11360. 1178–1184. Sands M, Kron MA, Brown RB. 1985. Pentamidine: A review. Rev Chang DD, Fisher RP, Clayton DA. 1987. Roles for a and Infect Dis 7: 625–634. RNA processing in the synthesis of mitochondrial displacement- Schmitt ME, Clayton DA. 1992. Yeast site-specific ribonucleoprotein loop strands. Biochim Biophys Acta 909: 85–91. endoribonuclease MRP contains an RNA component homologous Chu S, Archer RH, Zengel JM, Lindahl L. 1994. The RNA of RNase to mammalian RNase MRP RNA and essential for cell viability. MRP is required for normal processing of ribosomal RNA. Proc Genes & Dev 6: 1975–1985. Natl Acad Sci 91: 659–663. Schmitt ME, Clayton DA. 1993. Nuclear RNase MRP is required for Dichtl B, Stevens A, Tollervey D. 1997. Lithium toxicity in yeast is due correct processing of pre-5.8S rRNA in Saccharomyces cerevisiae. to the inhibition of RNA processing enzymes. EMBO J 16: 7184– Mol Cell Biol 13: 7935–7941. 7195. Schmitt ME, Clayton DA. 1994. Characterization of a unique protein Gill T, Cai T, Aulds J, Wierzbicki S, Schmitt ME. 2004. RNase MRP component of yeast RNase MRP: An RNA-binding protein with cleaves the CLB2 mRNA to promote cell cycle progression: Novel a zinc-cluster domain. Genes & Dev 8: 2617–2628. method of mRNA degradation. Mol Cell Biol 24: 945–953. Shevchenko A, Wilm M, Vorm O, Mann M. 1996. Mass spectrometric Gill T, Aulds J, Schmitt ME. 2006. A specialized processing body that sequencing of proteins silver-stained polyacrylamide gels. Anal is temporally and asymmetrically regulated during the cell cycle in Chem 68: 850–858. Saccharomyces cerevisiae. J Cell Biol 173: 35–45. Starck SR, Roberts RW. 2002. Puromycin oligonucleotides reveal Glick BS, Pon LA. 1995. Isolation of highly purified mitochondria steric restrictions for ribosome entry and multiple modes of from Saccharomyces cerevisiae. Methods Enzymol 260: 213–223. translation inhibition. RNA 8: 890–903. Gold HA, Topper JN, Clayton DA, Craft J. 1989. The RNA processing Stohl LL, Clayton DA. 1992. Saccharomyces cerevisiae contains an enzyme RNase MRP is identical to the Th RNP and related to RNase MRP that cleaves at a conserved mitochondrial RNA RNase P. Science 245: 1377–1380. sequence implicated in replication priming. Mol Cell Biol 12: Guerrier-Takada C, Gardiner K, Marsh T, Pace N, Altman S. 1983. 2561–2569. The RNA moiety of is the catalytic subunit of the Sun T, Zhang Y. 2008. Pentamidine binds to tRNA through non- enzyme. Cell 35: 849–857. specific hydrophobic interactions and inhibits aminoacylation and Hong EL, Balakrishnan R, Dong Q, Christie KR, Park J, Binkley G, translation. Nucleic Acids Res 36: 1654–1664. Costanzo MC, Dwight SS, Engel SR, Fisk DG, et al. 2008. Gene Thiel CT, Horn D, Zabel B, Ekici AB, Salinas K, Gebhart E, Ontology annotations at SGD: New data sources and annotation Ruschendorf F, Sticht H, Spranger J, Muller D, et al. 2005. Severely methods. Nucleic Acids Res 36: D577–D581. incapacitating mutations in patients with extreme short stature Karwan R, Bennett JL, Clayton DA. 1991. Nuclear RNase MRP identify RNA-processing endoribonuclease RMRP as an essential processes RNA at multiple discrete sites: Interaction with an cell growth regulator. Am J Hum Genet 77: 795–806. upstream G box is required for subsequent downstream cleavages. Thiel CT, Mortier G, Kaitila I, Reis A, Rauch A. 2007. Type and level Genes & Dev 5: 1264–1276. of RMRP functional impairment predicts phenotype in the Kikovska E, Svard SG, Kirsebom LA. 2007. Eukaryotic RNase P RNA cartilage hair hypoplasia-anauxetic dysplasia spectrum. Am J mediates cleavage in the absence of protein. Proc Natl Acad Sci Hum Genet 81: 519–529. 104: 2062–2067. Venema J, Tollervey D. 1999. Ribosome synthesis in Saccharomyces Liu Y, Tidwell RR, Leibowitz MJ. 1994. Inhibition of in vitro splicing cerevisiae. Annu Rev Genet 33: 261–311. of a group I intron of Pneumocystis carinii. J Eukaryot Microbiol 41: Vioque A. 1989. Protein synthesis inhibitors and catalytic RNA. Effect 31–38. of puromycin on tRNA precursor processing by the RNA Lygerou Z, Allmang C, Tollervey D, Seraphin B. 1996. Accurate component of Escherichia coli RNase P. FEBS Lett 246: 137– processing of a eukaryotic precursor ribosomal RNA by ribonu- 139. clease MRP in vitro. Science 272: 268–270. Woodhams MD, Stadler PF, Penny D, Collins LJ. 2007. RNase MRP Mann H, Ben-Asouli Y, Schein A, Moussa S, Jarrous N. 2003. and the RNA processing cascade in the eukaryotic ancestor. BMC Eukaryotic RNase P: Role of RNA and protein subunits of Evol Biol (Suppl 1) 7: S1–S13.

www.rnajournal.org 9 Downloaded from rnajournal.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press

Comparison of mitochondrial and nucleolar RNase MRP reveals identical RNA components with distinct enzymatic activities and protein components

Qiaosheng Lu, Sara Wierzbicki, Andrey S. Kraslinikov, et al.

RNA published online January 19, 2010

P

License

Email Alerting Receive free email alerts when new articles cite this article - sign up in the box at the Service top right corner of the article or click here.

To subscribe to RNA go to: http://rnajournal.cshlp.org/subscriptions

Copyright © 2010 RNA Society